RNA is central to a variety of biological processes including transcription, splicing, translation, gene expression, development, and cell division. It is therefore of interest to understand how RNA folds into the structures necessary to carry out these functions. It is also important to understand how the structures of native RNA and RNA folding intermediates regulate critical biological processes including activation of the interferon-induced anti-viral agent protein kinase PKR. This proposal involves studying RNA folding events that occur during transcription, and understanding their similarities and differences to refolding events that occur upon addition of divalent ions. The importance of transcriptional pausing with the authentic template and polymerase will be investigated. In addition, the ability of non-native, or alternative pairings, to inhibit and, in selected cases, stimulate the folding of the catalytic RNA from hepatitis delta virus (HDV) will be studied. The influence of external factors, such as increased ionic strength and hepatitis delta antigen protein (HDAg), on resolution of alternative pairings will be systematically investigated. The extent to which these RNA folding states can activate PKR will be investigated as well. A recently discovered novel small RNA motif that activates PKR will be studied mechanistically. These studies will be carried out with biochemical and biophysical techniques, including PKR activation and rapid-quench RNA cleavage kinetics; stopped-flow fluorescence and absorbance kinetics; thermodynamic measurements; and RNA structure mapping. It is anticipated that these results may impact upon several areas relevant to human health including understanding replication of HDV, which increases the virulence of hepatitis B virus (HBV) infections, and regulation of PKR protein, which mediates part of the human viral defense mechanism. In addition, these studies may help uncover new roles for the kinase in vivo. Results are expected to be of fundamental interest to the RNA folding community and may impact the understanding of RNA folding in other biologically relevant RNA and RNA-protein systems including catalytic RNAs and the ribosome.